Monoclonal antibody

ABSTRACT

Specific antibody of the invention is capable of differentiating between malignant and benign tumors, and is obtained by culturing hybridoma cells prepared from tumor cells treated with a certain anti-tumor substance.

This is a continuation of application Ser. No. 07/251,980, filed on Sep.28, 1988, which was abandoned upon the filing hereof which is Rule 62continuation of Ser. No. 06/684,262 filed Dec. 20, 1984, now abandoned.

FIELD OF THE INVENTION

The present invention relates to an antibody against a trace level ofantigen produced by the cells of a specific hybridoma, as well as aprocess for producing such antibody. More particularly, the inventionrelates to a specific antibody that is capable of differentiatingbetween malignant and benign tumors and which is obtained by sensitizingan animal with tumor cells which have been treated with an anti-tumorsubstance selected from the group consisting of cytokine and lymphokine,extracting the spleen from the animal, fusing the cells of the spleenwith hybridoma-producing, infinitely growing cells, cloning the fusedcells and finally culturing the cloned hybridoma cell line. Theinvention also relates to a process for producing such antibody. Theantibody of the present invention can be used for clinical purposes suchas diagnosis and treatment of human tumor cells or tissues.

PRIOR ART

In 1975, Kohler and Milstein prepared from a hybridoma a monoclonalantibody acting on a specific antigen (the monoclonal antibody ishereunder abbreviated as MoAb), the hybridoma being prepared by fusingmouse myeloma cells with the spleen cells of a mouse immunized withsheep red blood cells. Their experiment provided a basic method forpreparing MoAb (see Nature, 256: 495-497, 1975). Following the study ofKohler and Milstein, many researchers made attempts at preparing varioustypes of MoAb.

Two major advantages of the method for preparing MoAb by the fusion ofcells are the absence of the need for purifying the target antigen andthe possibility of producing a homogeneous antibody (MoAb) in largequantities. Because of these advantages, the potential importance ofMoAb will not be limited to basic research work; it is expected toprovide an effective tool in clinical fields (e.g. diagnosis andtreatment of cancers and other intractable diseases) by producingantibodies specific to malignant tumors. Particularly active efforts arebeing made in the field of tumor immunology wherein researchers areusing the cell fusion technique to prepare a wide variety of MoAbshaving specificity for tumor cells such as melanoma, colon cancer, andleukemic cells (see Proc. Natl. Acad. Sci. US. 75: 3405-3409, 1978,ibid. 76: 2927-2931, 1979, ibid. 77: 6114-6118, 1980, ibid. 76:1438-1442, 1979, Curr. Top. Microbiol. Immunol. 81: 164-169, 1978).

However, most of the MoAbs developed so far are cross-reacting andrecognize not only the target malignant tumor cells but also other cellssuch as benign tumor cells. Therefore, these MoAbs may show "falsepositiveness" when used in clinical applications such as diagnosis andtreatment of malignant tumors. In order to avoid this problem, it isnecessary to prepare an antibody against an antigen which is specificonly for the target malignant tumor cells. Several types of cancer havebeen found to contain tumor-associated antigens such as melanomaassociated antigen (MAA), α-feto protein (AFP), carcino embryonicantigen (CEA) and spleen cancer associated antigen (Science, 212: 53-55,1981), and attempts are being made to prepare MoAbs against theseantigens. One of the most advanced fields is the analysis of antigens inmelanoma, and clinical studies are being made on the MoAbs against theseantigens (see Melanoma-Antigens and Antibodies, ed. R. A. Reisfeld andS. Ferrone, Plenum Press, N.Y., 1982), but no MoAb has been obtainedthat is completely free from cross-reaction with benign tumors such asmelanocytes.

Usually, malignant tumor cells contain a very small amount of a specificantigen and the level of their antigenicity is so low that it isdifficult to obtain an antibody against this specific antigen bydirectly immunizing an animal with these tumor cells. In an attempt toobtain antibodies having specific reactivity, tumor cells with which toimmunize animals were treated with formalin or irradiated with UVradiation or X-rays so that the antigenicity of the tumor-specificantigen is sufficiently increased to obtain the desired MoAb (seeMelanoma-Antigens and Antibodies, ed. R. A. Reisfeld and S. Ferrone,Plenum Press, N.Y., 1982), but no satisfactory results have beenobtained by this approach.

As a result of various studies conducted with a view to solving theseproblems, the present inventors have found that by treating tumor cellswith an anti-tumor cytokine or lymphokine, preferably gamma-interferon(hereunder abbreviated as INF-γ) before they are used to immunize ananimal, the antigenicity of a previously unknown antigen that is presentin a trace level and which is specific to those tumor cells can beincreased to such a degree that an antibody specific to that antigen canbe produced in the animal. The inventors have also found that anantibody capable of differentiating between malignant and benign tumorscan be prepared from said antigen. These findings have led to theaccomplishment of the present invention.

Interferons (hereunder abbreviated as IFN) are known to have severalbiological functions such as anti-viral activity, inhibition of cellgrowth and anti-tumor activity. It is also known that some interferonsincrease the antigenicity of some common tissue-compatible antigens inan in vitro system, such as human leukocyte antigen (HLA), β₈₂-microglobulin and melanoma associated antigen (MAA). While many reportshave been published to show that α- and β-IFNs increase the antigenicityof Class I antigens (e.g. HLA-A and -B) in certain types of cells (seeProc. Natl. Acad. Sci. US. 75: 6215-6219, 1978, ibid. 79: 3265-3269,1982, ibid. 79: 3082-3086, 1982 and Eurp. J. Immunol. 9: 446-449, 1979),no report has been published as to whether IFN-γ has a similar activity.

SUMMARY OF THE INVENTION

In the course of the studies that led to the accomplishment of thepresent invention, the inventors treated malignant melanoma cells withIFN-γ and sensitized a mouse with the treated cells. Spleen cellsisolated from the sensitized mouse were then fused with mouse myelomacells, and the fused cells were cloned to obtain hybridomas. Thehybridomas were found to be capable of producing antibodies having anunusually high specific reactivity. A probable reason for the productionof such highly specific antibodies would be that the direct anti-tumoractivity of IFN-γ causes a certain change in the treated tumor cells toexhibit antibody-producing stimulation, and the animal sensitized withthese tumor cells produced a sufficient amount of specific antibody.Therefore, it is expected that substances other than IFN-γ that havedirect anti-tumor activity such as lymphokine (e.g. lymphotoxin, LT) andcytokine (e.g. tumor necreosis factor, TNF) would be useful in inducingtumor-specific antigens. It is also expected that by treating tumorcells other than melanoma cells with lymphokine or cytokine, previouslyundiscovered antigens can be induced, and useful antibodies against theinduced antigens, such as one that reacts specifically with the targettumor cells, can be created by the cell fusion technique.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the analysis of the antigen induced by IFN-γ treatment.

DETAILED DESCRIPTION OF THE INVENTION

The first stage of preparing the novel monoclonal antibody is to treattumor cells such as melanoma cells Colo 38 with cytokine or lymphokine,preferably with IFN-γ (e.g. IFN-γ produced by E. coli created byrecombinant DNA technology). The concentration of the treating agent andthe duration of the treatment of the tumor cells are such that they donot inhibit, or inhibit only slightly, the viability of the tumor cellsbut are sufficient to cause a certain stimulation in said cells.Suitable levels of concentration and duration of treatment may bedetermined empirically by those skilled in the art for each case. Asguide FIGURE, melanoma cells Colo 38 may be treated with 250-500U/ml ofIFN-γ for a period up to 96 hours, preferably about 72 hours. Othertreatment conditions such as temperature and pH may be those which areconventionally used in cell cultivation.

In the next stage, the tumor cells treated under the conditions shownabove are used to sensitize an animal by a standard method. Spleen cellsare isolated from the immunized animal and are fused withhybridoma-producing, infinitely growing cells. The fused cells arecloned to obtain a hybridoma that produces a monoclonal antibody thatrecognizes specifically the target malignant tumor. The sequence of thishybridoma production is already known and may comprise the followingsteps. About 5×10⁶ treated tumor cells are suspended in 0.2 ml of PBS(phosphate-buffered saline) and the suspension is injected into theabdominal cavity of a BALB/c mouse several times (preferably threetimes) at intervals of 7 to 10 days, thereby immunizing the animal.About three days after the final immunization, spleen cells extractedfrom the mouse are fused with mouse myeloma cells with the aid of afusing agent such as polyethylene glycol. The hybridoma is selectivelycultured in a HAT medium (RPMI 1640+10% serum; with hypoxanthine,aminopterin and thymidine) by a conventional method, and the supernatantof the culture is subjected to primary screening by the ELISA(enzyme-linked-immunosorbent assay) method for its reactivity with thetarget cells. The hybridoma found positive in the primary screening ispreferably sub-cloned by the limited dilution method to form amonoclone.

The sub-class of the monoclonal antibodies produced by the clonedhybridoma may be identified by an immunological technique. A commercialmouse globulin identifying kit may be conveniently used to identify thesub-class of mouse-derived MoAbs. Analysis of the antigen correspondingto the antibody of the present invention is performed by the followingprocedure: the solubilized protein from tumor cells labelled with aradioisotope is reacted with the antibody, and the antigen bound withsaid antibody is dissociated with β-mercaptoethanol, and subsequentlysubjected to SDS polyacrylamide gel electrophoresis (SDS PAGE). Thereactivity of the antibody with the tissue of the target tumor may beexamined by first fixing the extracted tumor tissue with acetone,staining the fixed tissue by the ELISA method, and observing the stainedtissue under a microscope.

As shown above, the essence of the present invention is to use asubstance having direct anti-tumor activity such as lymphokine,cytokine, or, preferably, INF-γ in preparing an antibody that recognizesa tumor-specific antigen which has been induced in an amount sufficientto exhibit antibody-producing stimulation. The so prepared antibodyreacts only with the desired malignant tumor cells and recognizes benigncells almost negligibly. It is also possible to obtain by the presentinvention an antibody that is highly reactive with the tissue of amalignant tumor extracted from the patient, as well as with the INF-γtreated tumor cells used as the antigen, and untreated tumor cells.

In the current diagnosis of cancer diseases, especially melanomadiseases, malignant tumors are distinguished from being tumorsexclusively by experienced doctors. As will be readily understood tothose skilled in the art, any paramedical technician having ordinaryknowledge can easily distinguish malignant tumors with the aid of theantibody of the present invention. This will therefore provide aneffective tool in the treatment of cancer diseases.

The advantages of the present invention will become more apparent byreading the following working examples, to which the scope of theinvention is by no means limited. It should be understood that theprocess of antibody production according to the present invention is notlimited to the preparation of an antibody specific to melanoma cells asillustrated in the following examples but that said process is alsoapplicable to the production of antibodies specific to other malignanttumor cells.

EXAMPLE 1) Treating melanoma cells Colo 38 with IFN-γ

Cultured melanoma cells Colo 38 (1×10⁵ cells/ml), as well as human IFN-γ(250U/ml and 500U/ml) isolated from transformed E. coli cells that wereconstructed by recombinant DNA technology, were cultivated in a medium(containing 10% calf serum and gentamicin, 1 μg/ml) for 96 hours at 37°C. As a control, the same melanoma cells were cultured under the sameconditions except that no IFN-γ was present. At 24-hr intervals, thenumber of living cells in each culture was counted by staining withTrypan Blue. A similar experiment was conducted using a higherconcentration of Colo 38 (6×10⁵ cells/ml) and a period of 72 hours. Whenthe initial concentration of Colo 38 was 1×10⁵ cells/ml, the cell growthwas inhibited by the presence of IFN-γ (cell number was about 60% of theuntreated cells after 72-hr culture in the presence of 250 U/ml ofIFN-γ), but no morphological changes were observed in the cells under amicroscope. When the initial concentration of Colo 38 was increased to6×10⁵ cells/ml, the percent survival was kept about 80% in the presenceof 250 U/ml of IFN-γ but a gradual decrease with time was observed atthe concentration of 500 U/ml.

Since the purpose of treating melanoma cells with IFN-γ was to inhibittheir growth and cause a certain stimulation of the membrane on the cellsurface while maintaining their survival as high as possible, thefollowing experiments were conducted by using 250U/ml of IFN-γ andlimiting the duration of IFN-γ treatment to 72 hr.

2) Immunization of mouse and hybridoma preparation

Melanoma cells (Colo 38, 10⁵ cells/ml) were cultured at 37° C. for 72hours in RPMI-1640 medium (with 10% calf serum and 1 μg/ml gentamycin)containing 250U/ml of human IFN-γ that was prepared by DNA recombinanttechnology. The cells were collected by centrifuge. After washing withPBS (phosphate buffered saline), 5×10⁶ cells were suspended in 0.2 ml ofPBS, and the suspension was injected into the abdominal cavity of aBALB/c mouse three times at intervals of 10 days.

Three days after the final immunization, the spleen was extractedaseptically from the mouse and dispersed into individual cells, whichwere then washed twice with serum free RPMI-1460. The number of spleencells that could be extracted from one mouse was in the range of1.2-1.4×10⁸. The spleen cells were fused with 2×10⁷ mouse myeloma cells(P3×63 Ag8 - 653) by treatment in RPMI-1640 containing 40% polyethyleneglycol 1000 for 5 minutes. After washing with RPMI-1640 containing 10%calf serum, the fused cells were suspended to give a concentration of5×10⁵ myeloma cells per ml, and the suspension was incubated for 24hours in the presence of 5% CO₂. The culture medium was replaced by aHAT medium (RPMI-1640 containing 10% calf serum, 10⁻⁴ M hypoxanthine,4×10⁻⁷ M aminopterin and 1.6×10⁻⁵ M thymidine), and the culture wascontinued on a microplate having 96 wells. Ten days later, the culturewas transferred into an aminopterin-free HAT medium (HT medium), and theculture was continued for an additional 14-21 days. The supernatant ofthe cell culture obtained was subjected to primary hybridoma screeningby the ELISA method.

3) Hybridoma screening and cloning 3-1) Screening by the ELISA method

Colo 38 cells treated with IFN-γ and those untreated with IFN-γ wereused as the target cells. A cell suspension containing 10⁵ target cellsin 50 μ1 of PBS and 50 μ1 of the culture supernatant of the hybridomawere incubated at 4° C. for 1 hour. Thereafter, the unadsorbedantibodies were washed away with Hanks solution containing 1% BSA(bovine serum albumin). The culture was reacted for 30 minutes with asecondary antibody which was a peroxidase labelled sheep anti-mouseantibody (product of Cappel Inc.) and washed with PBS. The colordevelopment of o-phenylenediamine was analyzed by a multiscanner(product of Titertech Inc.) at 405 nm. The results were estimated basedon the measured absorbancy as follows: 0-0.3 (-), 0.3-0.5 (±), 0.5-1.0(+) and >1.0 (++).

The 168 clones obtained by the HAT selection in 2) were checked fortheir reactivity with IFN-γ treated Colo 38 and with untreated Colo 38;whereby 34 clones had absorbance values of 0.3 or more. In order tocheck for the existence of anti-HLA antibody, 9 out of the 34 cloneswere subjected to the ELISA method with a view to determining theircross-reactivity with LG-2 cells of Burkitt's lymphoma B treated withIFN-γ and those untreated with IFN-Y (the LG-2 cells are positive toHLA-A, B and HLA-D_(R)). Five out of the nine clones tested werereactive with LG-2 irrespective of their treatment with IFN-γ. Theremaining four clones produced antibodies that did not react with LG-2but reacted with Colo 38.

3-2) Screening by histological method

The clones selected in 3-1) were checked for their reactivity withtissue specimens. Melanoma tissues obtained by biopsy or surgicaloperation were sliced to a 4-μm thick section which was fixed withacetone and overlaid with the supernatant of the culture of a testclone. The specimen was left to stand for 30 minutes at roomtemperature. The specimen was washed with cooled PBS for 5 minutes andreacted with peroxidase labelled anti-mouse serum for 30 minutes. Theunreacted mouse serum was washed with cold PBS, then with hydrogenperoxide (0.1-1%). The degree of brown staining of the sample wasexamined under a microscope.

The supernatant of the culture of 34 clones that were found positive in3-1) were also checked for their reactivity with melanoma tissues by thesame procedure. The results were as follows: two clones (CL-208 andCL-308) were extremely reactive; five clones (CL-203, CL-207 and threeothers) were strongly reactive; and ten clones had moderate reactivity.Although the correlation between the reactivity with cultured Colo 38cells and that with the extracted tissues was not clear enough, theclones that strongly reacted with the culture of IFN-γ treated Colo 38cells had a tendency to strongly react with the extracted tissues also.

It is interesting to note that the supernatants of the culture of twoclones (CL-203 and CL-207) that exhibited strong reactivity both withcultured Colo 38 cells and with extracted tissues did not react with abenign tumor (melanocyte), and their reactivity with LG-2 cells wasalmost negligible. This indicates that the antibody produced by theclones (hybridomas) designated CL-203 and CL-207 are useful not only indistinguishing a malignant tumor (melanoma) from a benign tumor(melanocyte) but also in treating melanoma diseases. CL207 SBM296 wasdeposited at the Fermentation Research Institute, 1-3, Higashil1-chrome, Tsukuba-shi, Ibaraki-Ken, 305 Japan, on Nov. 10, 1987. Thedeposit has been accorded accession No. FERM BP-1567.

3-3) Preparation of monoclonal hybridoma

The positive clones selected in 3-1) and 3-2) were subcloned tomonoclonal antibodies by the limited dilution method in the followingmanner. The hybridoma was diluted to give a concentration of not morethan 10 cells per ml, and the dilution was distributed among 96 wells ina microplate so that each well contained 0.1 ml of the dilution. Themicroplate was subjected to incubation in HAT medium.

As feeder layer cells, 2×10⁵ ml of spleen cells of BALB/c mice wereused.

4) Antigen analysis

The types of antigens that could be recognized by the antibodiesproduced from several clones of the hybridoma prepared in 3) weredetermined both by the immunological precipitation method and byexamining the clones' reactivity with Colo 38 cells being cultured inthe presence of IFN-γ.

5 4-1) Analysis by the immuno-precipitation method

Cultured Colo 38 cells whose protein had been labelled with ³⁵S-methionine were collected and the protein that solubilized with asurfactant (Renex 30) was used as an antigen. The supernatant of acultured hybridoma (50 μ1) was reacted at 4° C. for 30 minutes withprotein A-cephalose CL4B (50 μ1, Pharmacia Labs., Inc.) to which rabbitanti-mouse antibody had been bound. To the reaction mixture, 10 μ1 ofthe 35S-methionine-labelled antigen was added. The resulting mixture wasleft to stand for 60 minutes, then washed five times with PBS bycentrifuge. The finally obtained complex (precipitate) was boiled for 2minutes in 100 μ1 of 1% SDS containing 1 mM β-mercaptoethanol.Thereafter, the mixture was subjected to 3-15% SDS-PAGE(SDS-polyacrylamide gel electrophoresis) to analyze the occurrence ofprotein bands labelled with ³⁵ S-methionine.

The clones of the present invention, i.e., CL-203 and CL-207, as well astheir respective subclones CL-203-4 and CL-207-14, which producedantibodies reacting with melanoma but not with a benign tumor(melanocyte), had protein bands at about 100 kd. On the other hand,CL-208 and CL-306, which produced antibodies outside the scope of thepresent invention and which reacted strongly not only with melanomacells and tissues but also with the benign melanocyte, had protein bandsat ca. 44 kd and ca. 12 kd, respectively. It is therefore clear that theantibodies according to the present invention recognize specificantigens that differ from those produced by CL-208 and CL-306.

The immunoglobulin subclass of the antibody produced by CL-203 andCL-207 was identified as IgGl, using a mouse immunolobulin subtypeidentification kit (product of Boehringer Mannheim Biochemicals,Indianapolis, U.S.A.).

4-2) Analysis of reactivity with antigens induced by IFN-γ treatment

A check was made as to whether the antibodies produced by CL-203 andCL-207, as well as by their subclones CL-203-4 and CL-207-14, couldrecognize specifically the antigens induced in IFN-γ treated Colo 38cells. Colo 38 cells were incubated for 4 days in RPMI-1640 medium(containing 10% calf serum) containing 250U/ml of IFN-γ and in IFN-γfree RPMI-1640 medium. The change in the amounts of the antigensrecognized by the antibodies produced by CL-203 and CL-207 was measuredby the ELISA method. The same experiment was conducted with two otherclones, i.e., CL-212 as prepared in this Example which produces anantibody outside the scope of the present invention which recognizesColo 38 cells strongly, and CL-611, also prepared in this Example, whichrecognizes Colo 38 cells moderately. The results are shown in FIG. 1,from which one can see that the amount of the antigen recognized by theantigens from CL-203 and CL- 207 that were induced in IFN-γ treated Colo38 cells increased with incubation time whereas no change was observedin the amount of the antigen recognized by the antigens produced fromCL-212 and CL-611. This shows that, as a result of the treatment withIFN-γ, antigens were induced in sufficient amounts to exhibit strongantigenicity. It can be concluded that CL-203 and CL-207 are clones thatproduce antibodies against the so induced antigens.

The foregoing description was made referring only to the antibodyproduced by hybridoma cells. However, in a practical application of thepresent invention, a malignant tumor specific antigen induced in tumorcells such as melanoma that have been treated with an anti-tumorsubstance such as gamma interferon can be isolated, purified, andadministered to an animal, whereupon an antibody specific to themalignant tumor will be produced in vivo. The antibody obtained fromthis animal can be used in the diagnosis or treatment of malignanttumors as effectively as the antibody produced by hybridoma cellsaccording to the present invention.

What is claimed is:
 1. A hybridoma cell line having all the identifyingcharacteristics of the hybridoma cell line having F.R.I. Accession No.FERM BP-1567, wherein said hybridoma cell line produces a monoclonalantibody which immunologically binds an antigen of about 100 kD which isinduced or enhanced on the surface of Colo 38 melanoma cells, saidmelanoma cells having been previously contacted with gamma interferon,and said antibody being non-reactive with any other antigens existing inColo 38 melanoma cells.
 2. The hybridoma cell line of claim 1 havingF.R.I. Accession No. FERM BP-1567.
 3. A monoclonal antibody produced bythe hybridoma of claim
 1. 4. A monoclonal antibody produced by thehybridoma of claim 2.